Cell polarization is one of the most fundamental organizational aspects of life, from single-celled organisms such as E. coli and budding yeast up to the wiring of the human brain. Cell polarization is required for the generation of diverse cell types from stem cells, for morphogenesis, directional motility, antigen presentation, and axon guidance;and the loss of cell polarity is a critical step in cancer progression. The wide interest in cell polarity has pushed rapid progress in understanding the scope and complexity of the polarization machinery. However, the fundamental question of how the polarity proteins operate to organize cell structure remains largely unanswered. This proposal builds on progress over the last funding period to address these questions. Our objective is to understand the connections between the PAR polarity proteins and small GTPases. We will focus on MDCK epithelial cell polarization in 3D cultures, and on dendritic spine morphogenesis in hippocampal neurons - two tractable systems in which the PAR proteins have defined but distinct roles. The specific aims are as follows: 1. Identify the molecular mechanisms through which PAR-3, PAR-6, and aPKC organize the apical domain (which we term a "patch") during polarized cyst formation of MDCK cells. Using live cell imaging, we will investigate the earliest steps in polarization, which occur during the initial cell division of cells grown in 3D cultures. We will test a new model in which cytokinetic events are proposed to generate the landmark for apical patch assembly. 2. Determine the role of CDC42 in assembly of the apical patch, and identify the CDC42 GEF and GAP that are necessary for cell polarization. RNAi screens are being used to identify the GEF and GAP. Based on preliminary data from a GEF RNAi screen, we propose that a CDC42-GEF called Tuba drives recruitment of PAR-6/aPKC and apicalization. 3. Identify the mechanisms by which PAR-3 controls TIAM1 function in neurons;and determine how PAR- 6, independently of PAR-3, regulates dendritic spine morphogenesis via the Rho GTPase. We will determine the molecular basis for activation of p190 RhoGAP by PAR-6/aPKC. The mechanism by which PAR-6 regulates synaptic activity in hippocampal neurons will also be investigated. Together, these studies will provide new insights into the fundamentally important process of cell polarization, and into the ways by which the PAR polarity proteins exhibit context-specific behavior to control different aspects of cell morphology. PUBLIC HEALTH RELEVANCE: Over 90% of all human cancers originate from epithelial cells. A key property of epithelial cells is that they adhere to one another to form sheets in which the upper surface (which faces the environment) is functionally different from the bottom surface. Cancer progression involves the temporary loss of this cell-cell adhesion, and of up-down polarity. Thus, to understand human cancer, and to identify new chemotherapeutic targets, it is important to find out how epithelial cells polarize. Remarkably, the same process that helps these cells stick to one another is also essential for making the contacts between nerve cells in the brain, which enable us to think and store memories. This grant investigates the detailed molecular mechanisms that enable epithelial cells and nerve cells to polarize and form junctions, and will provide new insights into these mechanisms.